The central goal of this project is to understand the role of cellular immunity in infections caused by LPS-smooth strains of P. aeruginosa. These strains are significant pathogens for hospitalized patients and wearers of extended-use contact lenses. Recent evidence suggests that during the course of lung and eye infections, P. aeruginosa enters epithelial cells via the cystic fibrosis transmembrane conductance regulator. To study the immune response to the intracellular base of infection, live, attenuated P. aeruginosa strains having an unmarked deletion of the aroA gene have been constructed. Preliminary studies reveal that intranasal (IN) immunization with one such mutant protects against keratitis or lethal pneumonia in murine models. Splenic T cells from immunized mice can kill intracellular P. aeruginosa. Interestingly, passive transfer of antiserum is more protective against corneal infections than lung infections. The candidate will further define the cellular immunity to aroA deletion mutants of P. aeruginosa and test the following hypothesis: The protective efficacy of IN immunization with live, attenuated P. aeruginosa vaccine strains requires both humoral and cellular immune effecters, and the relative contribution of each effecter towards protection is different in different sites of infection. In Aim 1, the cellular immune effecters elicited by aroA deletants of P. aeruginosa will be delineated using immunomagnetic depletion coupled with assays for intracellular bacterial killing, T cell proliferation, and cytolysis. In Aim 2, P. aeruginosa specific T cell clones from IN-immunized mice will be derived and characterized. The candidate seeks an intensive, formal, mentored training to provide the necessary intellectual and technical tools to achieve independence as a scientist. As a specialist in pediatric critical care and infectious diseases, his long-term goal is to develop effective immune-based interventions to prevent or ameliorate the consequences of P. aeruginosa infections.